Pullulanases are enzymes found useful in numerous industrial applications, especially in the food and beverage industries. Pullulanases are starch debranching enzymes and are effective in the debranching of starch hydrolyzates (useful in conditioning dough), the debranching of β-limit dextrans (useful in the brewing of beer and ales) and in the production of sugar syrups from corn, potato, wheat, manioc and rice, for example. Pullulanases are enzymes classified in EC 3.2.1.41 and such enzymes are characterized by their ability to hydrolyze the α-1,6-glycosidic bonds in, for example, amylopectin and pullulan.
Pullulanases are the product of bacteria, especially of the genus Bacillus. The production of pullulanases for industrial use is not without problems. Pullulanases are quickly degraded by various proteases also produced by the bacteria thereby making the recovery of large quantities of pullulanase inefficient and expensive. Various persons in the field have devised methods to increase production by limiting the degradation of pullulanase in the culture. For example, we have previously shown that deletion of the AprL and Mpr genes (which expressed proteases) from a pullulanase production strain was necessary for the economical expression of active pullulanase. Still, the fermentation time is limited to 51-60 hours to limit proteolytic degradation and activity loss of the pullulanase product. Svendsen has designed pullulanase variants that alter the three dimensional conformation of the enzyme to increase the thermal stability of the enzyme or to change how the enzyme degrades its substrate (see, U.S. Pat. Nos. 6,350,599 and 6,838,257 as well as US application no. 2004/0082028).
More recently, we have shown that the timing of pullulanase degradation was determined with the following result: between 30 and 50 hours a partial clipping of the full length pullulanase molecule into truncated molecules lacking the N-terminal 98 and 102 amino acids, respectively, was observed. Clipping occurred N-terminally of glutamic acid residues E99 and E103, respectively, and could be visualized by HPLC. Surprisingly, the 1-98 and 1-102 truncated pullulanase molecules retain pullulanase activity and even more surprising, it was demonstrated that this activity is higher than that of the full-length pullulanase. After 51 hours, further degradation of pullulanase molecules resulted in activity drop that eventually abolished all activity.
Still, there is room for improvement in the design of pullulanase peptides and the nucleotide seqeunces that encode them. Therefore, what is needed are compounds and methods for the more efficient production of pullulanase by, for example, limiting proteolytic degradation, increasing fermentation titers or increasing pullulanase activity.